Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to a composition which comprises peptides derived from S- Arrestin (retinal arrestin, S-antigen, S-Ag).
• S- Arrestin receptor arrestin, S-antigen, S-Ag.
• the composition or peptides may be useful in the prevention and/or suppression of S-Ag autoimmunity, which is useful in the treatment and/or prevention of uveitis.
• Uveitis describes a group of diseases associated with inflammation of the uvea.
• the uvea is a region of the eye located between the sclera and the retina, and includes the iris, ciliary body and choroid.
• the uvea provides most of the blood supply to the retina.
• the associated diseases are not restricted to those affecting the uvea directly, and adjacent structures such as the retina, optic nerve, lens, vitreous and sclera can be affected in manifestations of uveitis.
• uveitis All forms of uveitis are characterised by an inflammatory cellular infiltrate, commonly visualised using a biomicroscope. In 2010, it was estimated that 285 million people were visually impaired; of these, 39 million were blind and it was approximated that 10% of the cases were due to uveitis. (Global data on visual impairments, The World Health Report, WHO (2010) http://www.who.int/blindness/GLOBALDATAFlNALforweb.pdf) Present treatments for uveitis include the use of glucocorticoid steroids and other immunosuppressive agents such as methotrexate.
• the present inventors have identified a number of peptides derived from S-Ag which may be useful in the prevention and/or treatment of uveitis.
• the present invention provides a peptide which comprises all or a portion of the following S-Ag derived peptides: KKKVIFKKISRDKSVTIYLGKKK (SEQ ID No. 15)
• the peptide is capable of binding to an MHC molecule in vitro and being presented to a T cell without antigen processing
• the present invention provides a composition comprising a plurality of peptides, including one of more peptides according to the first aspect.
• a peptide according to the first aspect or a composition according to the second aspect for use in supressing or preventing the production of T cells specific for S-Ag and/ or S-Ag autoantibodies in vivo.
• a peptide according to the first aspect or a composition according to the second aspect for use in treating or preventing uveitis in a subject.
• the present invention provides a method for suppressing or preventing the production of S-Ag autoantibodies in a subject, which comprises the step of administration of a peptide according to the first aspect, or a composition according to the second aspect, to the subject.
• the present invention provides a method for treating or preventing uveitis in a subject, which comprises the step of administration of a peptide according to the first aspect, or a composition according to the second aspect, to the subject.
• FIG. 1 Immunogenicity of HIP-1 15 in DR3 mice. Mice were immunized with 50 ⁇ g HIP- 115/CFA or PBS/CFA in the tail base. After 10 days, LN and spleens were harvested and cultured with 10 ⁇ g/ml and 25 ⁇ g/ml of HIP-1 15. Supernatants were collected after 72h. Cell activation was measured by IFN- ⁇ ELISA.
• A Immunogenicity in LN.
• B Immunogenicity in spleens. CFA, Complete Freund's Adjuvant; LN, lymph nodes.
• Figure 2 identification of apitopes within HIP-1 15.
• A DR3 mice were immunized with SAg and hybridomas were generated.
• 5x10 4 SAg-specific hybridoma cells were cultured with 5x10 4 fresh or fixed commercial APC (VAVY) cells. T cell proliferation was measured by IL-2 ELISA on supernatants collected after 48h. The graph represents the mean of duplicate measurements ⁇ SEM.
• B DR3 mice were immunized with HIP-115 and a T cell line was established. Ten days after immunization the LN and spleens were harvested and cultured with different concentrations of peptide (0, 1-1-2,5 and 5 Mg/ml); 2x10 6 cells/ml APC + 1x10 6 cells/ml CD4 T cells per well. At day 7 the cells were re-stimulated with the same concentrations of peptide.
• FIG. 3 identification of apitopes within HIP-241125.
• A DR3 mice were immunized with SAg and hybridomas were generated. 5x10 4 SAg-specific hybridoma cells were cultured with 5x10 4 fresh or fixed commercial APC (VAVY) cells. T cell proliferation was measured by IL-2 ELISA on supernatants collected after 48h. The graph represents the mean of duplicate measurements ⁇ SEM.
• B DR3 mice were immunized with HIP-241125+HIP-24DG and a T cell line was established.
• LN and spleens were harvested and cultured with different concentrations of peptide (0, 1-1-2,5 and 5 ⁇ g/ml); 2x10 6 cells/ml APC + 1x10 6 cells/ml CD4 T cells per well. At day 7 the cells were re-stimulated with the same concentrations of peptide.
• an APIPS test was done using commercial APC (VAVY). After 24h, antigen-induced T cell activation was measured by IFN- ⁇ ELISA and shown as IFN- ⁇ concentration (pg/ml). The graph represents the mean of duplicate measurements ⁇ SEM.
• LN lymph nodes
• APC Antigen presenting cell
• APIPS Antigen Processing Independent Presentation System.
• FIG. 4 Identification of apitopes within HIP-9FL.
• DR2 mice were immunized with HIP-9FL- KKK and a T cell line was established.
• Ten days after immunization the LN and spleens were harvested and cultured with different concentrations of peptide (0,1-1-2,5 and 5 ⁇ g/ml); 2x10 6 cells/ml APCs + 1x10 6 cells/ml CD4 T cells per well.
• the cells were re- stimulated with the same concentrations of peptide.
• an APIPS test was done using commercial APC (MGAR). After 24h, antigen-induced T cell activation was measured by IFN- ⁇ ELISA and shown as OD values.
• the graph represents the mean of duplicate measurements ⁇ SEM.
• LN lymph nodes
• APC Antigen presenting cell
• APIPS Antigen Processing Independent Presentation System.
• Figure 5 Identification of apitopes within HIP-17GN.
• DR3 mice were immunized with HIP- 17GN and a T cell line was established.
• Ten days after immunization the LN and spleens were harvested and cultured with different concentrations of peptide (0, 1-1-2,5 and 5 ⁇ g/ml); 2x10 6 cells/ml APC + 1x10 6 cells/ml CD4 T cells per well.
• At day 7 the cells were re- stimulated with the same concentrations of peptide.
• an API PS test was done using commercial APC (VAVY).
• the graph represents the mean of triplicate measurements ⁇ SEM.
• LN lymph nodes
• APC Antigen presenting cell
• APIPS Antigen Processing Independent Presentation System.
• FIG. 6 Identification of apitopes within HIP-12AK.
• DR3 mice were immunized with HIP- 12AK and a T cell line was established.
• Ten days after immunization the LN and spleens were harvested and cultured with different concentrations of peptide (0, 1-1-2,5 and 5 ⁇ g/ml); 2x10 6 cells/ml APC + 1x10 6 cells/ml CD4 T cells per well. At day 7 the cells were re- stimulated with the same concentrations of peptide.
• an APIPS test was done using commercial APC (VAVY). After 24h, antigen-induced T cell activation was measured by IFN- ⁇ ELISA and shown as IFN- ⁇ concentration (pg/ml).
• the graph represents the mean of duplicate measurements ⁇ SEM.
• LN lymph nodes
• APC Antigen presenting cell
• APIPS Antigen Processing Independent Presentation System.
• Figure 7 Ex vivo tolerisation protocol. Mice are injected subcutaneously in the flank with 0, 1 ⁇ g/ml, 1 ⁇ g/ml and 10 ⁇ g/ml peptide on days -15, -13 and -11 , followed by 3 injections of 100 ⁇ g/ml on days -8, -6 and -4 (escalating dose regime). On day 0, mice are immunized subcutaneously at the base of the tail with SAg/CFA or peptide/CFA. Mice are sacrificed 10 days after immunization to measure proliferation of LN cells and splenocytes upon SAg or peptide restimulation. LN, lymph nodes.
• FIG. 8 Ex vivo tolerance induction by HIP-1 15NE3-KKK apitope.
• DR3 mice are injected subcutaneously in the flank with 0,1 ⁇ g/ml, 1 ⁇ g/ml and 10 ⁇ g/ml HIP-1 15NE3-KKK on days -15, -13 and -11 , followed by 3 injections of 100 ⁇ g/ml on days -8, -6 and -4 (escalating dose regime).
• mice are immunized subcutaneously at the base of the tail with HIP- 115/CFA. Mice are sacrificed 10 days after immunization to measure proliferation of LN cells and splenocytes upon SAg or peptide restimulation.
• FIG. 9 Ex vivo tolerance induction by HIP-11 B apitope.
• DR3 mice are injected subcutaneously in the flank with 0,1 ⁇ g/ml, 1 ⁇ g/ml and 10 ⁇ g/ml HIP-11 B on days -15, -13 and -11 , followed by 3 injections of 100 ⁇ g/ml on days -8, -6 and -4 (escalating dose regime).
• mice are immunized subcutaneously at the base of the tail with HIP- 11 B/CFA. Mice are sacrificed 10 days after immunization to measure proliferation of LN cells and splenocytes upon SAg or peptide restimulation.
• FIG. 10 Ex vivo tolerance induction by HIP-24DG apitope.
• DR3 mice are injected subcutaneously in the flank with 0,1 ⁇ g/ml, 1 ⁇ g/ml and 10 ⁇ g/ml HIP-24DG on days -15, -13 and -11 , followed by 3 injections of 100 ⁇ g/ml on days -8, -6 and -4 (escalating dose regime).
• mice are immunized subcutaneously at the base of the tail with HIP- 24DG+HIP-24HM/CFA.
• Mice are sacrificed 10 days after immunization to measure proliferation of LN cells and splenocytes upon SAg or peptide restimulation.
• mice are sacrificed 10 days after immunization to measure proliferation of LN cells and splenocytes upon SAg or peptide restimulation.
• Data represent mean ⁇ SEM of the concentration values for the PBS-treated mice (black lines) and peptide-treated mice (color lines).
• IFN- ⁇ production expressed as IFN- ⁇ concentration (pg/ml).
• Two-way ANOVA was used to measure overall treatment effects on T cell activation and p-values are written in the graphs. Bonferonni post-hoc testing was used and significant differences are indicated in the graphs (* p ⁇ 0.05; **** p ⁇ 0.0001).
• LN lymph nodes.
• Figure 12 Ex vivo tolerance induction by HIP-17GN-KKK and HIP-17J-KKK apitopes.
• DR3 mice are injected subcutaneously in the flank with 0, 1 ⁇ g/ml, 1 ⁇ g/ml and 10 ⁇ g/ml HIP- 17GN-KKK or HIP-17J-KKK on days -15, -13 and -11 , followed by 3 injections of 100 ⁇ g/ml on days -8, -6 and -4 (escalating dose regime).
• mice are immunized subcutaneously at the base of the tail with HIP-17GN/CFA. Mice are sacrificed 10 days after immunization to measure proliferation of LN cells and splenocytes upon SAg or peptide restimulation.
• mice are injected subcutaneously in the flank with 0, 1 ⁇ g/ml, 1 ⁇ g/ml and 10 ⁇ g/ml HIP-12G1-KKK on days - 15, -13 and -11 , followed by 3 injections of 100 ⁇ g/ml on days -8, -6 and -4 (escalating dose regime).
• mice are immunized subcutaneously at the base of the tail with HIP- 12AK/CFA.
• Mice are sacrificed 10 days after immunization to measure proliferation of LN cells and splenocytes upon SAg or peptide restimulation.
• Data represent mean ⁇ SEM of the concentration values for the PBS-treated mice (black lines) and peptide-treated mice (color lines).
• IFN- ⁇ production expressed as IFN- ⁇ concentration (pg/ml).
• Two-way ANOVA was used to measure overall treatment effects on T cell activation and p-values are written in the graphs. Bonferonni post-hoc testing was used and significant differences are indicated in the graphs (** p ⁇ 0.01 ; *** p ⁇ 0.001 : **** p ⁇ 0.0001).
• LN lymph nodes.
• A Tolerisation against HIP-12G1-KKK and HIP-12G1 in LN.
• B Tolerisation against HIP-12G1-KKK in spleen.
• Figure 14 In vivo presentation protocol. Mice are immunized with peptide/CFA and a T cell line is established.
• FIG. 15 In vivo presentation of HIP-115 nested peptides in DR3 mice.
• DR3 mice were immunized with HIP-1 15/CFA and a T cell line was established. For this, ten days after immunization the LN and spleens were harvested and cultured with different concentrations of peptide (0, 1-1-2,5 and 5 Mg/ml); 2x10 6 cells/ml APC + 1x10 6 cells/ml CD4 + cells per well. At day 7 the cells were re-stimulated with fresh APC and the same concentrations of peptide. After 14 days, CD4 + cells were isolated from these cultures by magnetic bead isolation.
• CD4 + cells were co-cultured with CD11 c + (dendritic) cells isolated by magnetic bead isolation from spleens of DR3 mice s.c. injected with peptides HIP-1 15NE3, HIP-115NE3-KKK or PBS 2 hours before. After 48 hours, supernatant was collected and IFN- ⁇ production was measured by ELISA. Data are presented as the mean IFN- ⁇ concentration ⁇ SEM (One-way ANOVA with Dunn's multiple comparison, * p ⁇ 0.05, ** p ⁇ 0.01). LN, lymph nodes; APC, Antigen presenting cell; s.c. subcutaneous.
• Figure 16 In vivo presentation of HIP-24 nested peptides in DR3 mice.
• DR3 mice were immunized with HIP-241125+HIP-24DG/CFA and a T cell line was established. For this, ten days after immunization the LN and spleens were harvested and cultured with different concentrations of peptide (0,3-1.2-3 and 6 Mg/ml); 2x10 6 cells/ml APC + 1x10 6 cells/ml CD4 + cells per well. At day 7 the cells were re-stimulated with fresh APC and the same concentrations of peptide. After 14 days, CD4 + cells were isolated from these cultures by magnetic bead isolation.
• CD4 + cells were co-cultured with CD11 c + (dendritic) cells isolated by magnetic bead isolation from spleens of DR3 mice s.c. injected with peptides HIP-24DG, HIP-24HM2, HIP-24HM3 or PBS 2 hours before. After 48 hours, supernatant was collected and IFN- ⁇ production was measured by ELISA. Data are presented as the mean IFN- ⁇ concentration ⁇ SEM (One-way ANOVA with Dunn's multiple comparison, * p ⁇ 0.05). LN, lymph nodes; APC, Antigen presenting cell; s.c. subcutaneous.
• Figure 17 In vivo presentation of HIP-9FL nested peptides in DR2 mice.
• mice were immunized with HIP-9FL-KKK/CFA and a T cell line was established. For this, ten days after immunization the LN and spleens were harvested and cultured with different concentrations of peptide (0, 1-1-2,5 and 5 Mg/ml); 2x10 6 cells/ml APC + 1x10 6 cells/ml CD4 + cells per well. At day 7 and day 14, the cells were re-stimulated with fresh APC and the same concentrations of peptide. After 21 days, CD4 + cells were isolated from these cultures by magnetic bead isolation.
• CD4 + cells were co-cultured with CD11 c + (dendritic) cells isolated by magnetic bead isolation from spleens of DR2 mice s.c. injected with peptides HIP-9K1 , HIP-9K1-KKK or PBS 2 hours before. After 48 hours, supernatant was collected and IFN- ⁇ production was measured by ELISA. Data are presented as the mean IFN- ⁇ concentration ⁇ SEM (One-way ANOVA with Dunn's multiple comparison, * p ⁇ 0.05). LN, lymph nodes; APC, Antigen presenting cell; s.c. subcutaneous.
• FIG. 18 In vivo presentation of HIP-17GN nested peptides in DR3 mice.
• DR3 mice were immunized with HIP-17GN/CFA and a T cell line was established.
• the LN and spleens were harvested and cultured with different concentrations of peptide (0, 1-1-2,5 and 5 Mg/ml); 2x10 6 cells/ml APC + 1x10 6 cells/ml CD4 + cells per well.
• the cells were re-stimulated with fresh APC and the same concentrations of peptide.
• CD4 + cells were isolated from these cultures by magnetic bead isolation.
• CD4 + cells were co-cultured with CD11 c + (dendritic) cells isolated by magnetic bead isolation from spleens of DR3 mice s.c. injected with peptides HIP-17GN- KKK, HIP-17J-KKK or PBS 2 hours before. After 48 hours, supernatant was collected and IFN- ⁇ production was measured by ELISA. Data are presented as the mean IFN- ⁇ concentration ⁇ SEM (One-way ANOVA with Dunn's multiple comparison, * p ⁇ 0.05). LN, lymph nodes; APC, Antigen presenting cell; s.c. subcutaneous.
• peptide is used in the normal sense to mean a series of residues, typically L- amino acids, connected one to the other, typically by peptide bonds between the a-amino and carboxyl groups of adjacent amino acids.
• the term includes modified peptides and synthetic peptide analogues.
• the peptide of the present invention may be made using chemical methods (Peptide Chemistry, A practical Textbook. Mikos Bodansky, Springer-Verlag, Berlin.). For example, peptides can be synthesized by solid phase techniques (Roberge JY et al (1995) Science 269: 202-204), cleaved from the resin, and purified by preparative high performance liquid chromatography (e.g., Creighton (1983) Proteins Structures And Molecular Principles, WH Freeman and Co, New York NY). Automated synthesis may be achieved, for example, using the ABI 43 1 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.
• the peptide may alternatively be made by recombinant means, or by cleavage from a longer polypeptide.
• the peptide may be obtained by cleavage from the S-antigen protein, which may be followed by modification of one or both ends.
• the composition of a peptide may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure).
• the peptide may show various other characteristics which the peptide may show. For example, it is important that the peptide is sufficiently stable in vivo to be therapeutically useful.
• the half-life of the peptide in vivo may be at least 10 minutes, 30 minutes, 4 hours, or 24 hours.
• the peptide may also demonstrate good bioavailability in vivo.
• the peptide may maintain a conformation in vivo which enables it to bind to an MHC molecule at the cell surface without due hindrance.
• compositions and kits of the present invention may be all or a portion of the following S-Ag derived peptides:
• VIGLTFRRDLYFSRVQVYPPVG (SEQ ID No 3)
• compositions and kits of the present invention may be all or a portion of peptides shown in the Table below:
• HIP-115 HIP-115 MAASGKTSKSEPNHVIFKKISRDKSVTIYLGNRDYIDHVSQV 10
• the peptide is selected from:
• the peptide has at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to any one of SEQ ID NOs: 1 to 9.
• the peptide has at least 80%, 90%, 95%, 97% or 99% sequence identity to any one of SEQ ID NOs: 1 to 46.
• Sequence identity may be assessed by any convenient method. However, for determining the degree of sequence identity between sequences, computer programs that make multiple alignments of sequences are useful, for instance Clustal W (Thompson et al., (1994) Nucleic Acids Res., 22: 4673-4680).
• variants of the stated or given sequences are variants of the stated or given sequences, as long as the variant retains the functional activity of the parent i.e. the variants are functionally equivalent, in other words they have or exhibit an activity of the parent peptide as defined herein.
• variants may comprise amino acid substitutions, additions or deletions (including truncations at one or both ends) of the parent sequence e.g. of one or more e.g. 1 to 14 amino acids.
• amino acids are chemically derivatised, e.g. substituted with a chemical group.
• the peptides of the invention can comprise portions or fragments of SEQ ID NOs 1-46, provided that the peptide retains the required activity.
• Portions or fragments of SEQ ID NOs 1-46 may for example be from 6 to 14 residues in length, e.g. 6, 7, 8, 9, 10, 11 , 12 or 13 residues in length.
• the peptide of the present invention may comprise between 8 and 30 amino acids, for example 8 to 25 amino acids, 8 to 20 amino acids, 8 to 15 amino acids or 8 to 12 amino acids.
• the peptide of the present invention may thus be 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length.
• a peptide composition according to the invention may comprise the amino acid sequences according to the invention as described herein.
• the peptide composition comprises only the amino acid sequences according to the invention as described herein, i.e. it does not comprise additional peptides other than those according to the invention.
• the peptides of the invention may be formulated into the composition as neutral or salt forms.
• Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric and maleic acid. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine and procaine.
• T lymphocytes are capable of recognising epitopes of a protein antigen.
• APCs take up protein antigens and degrade them into short peptide fragments.
• a peptide may bind to a major histocompatibility complex (MHC) inside the cell and be carried to the cell surface.
• MHC major histocompatibility complex
• the peptide When presented at the cell surface in conjunction with an MHC molecule, the peptide may be recognised by a T cell (via the T cell receptor (TCR), in which case the peptide is a T cell epitope.
• TCR T cell receptor
• An epitope is thus a peptide derivable from an antigen which is capable of binding to the peptide-binding groove of an MHC molecule and being recognised by a T cell.
• the minimal epitope is the shortest fragment derivable from an epitope, which is capable of binding to the peptide-binding grove of an MHC class I or II molecule and being recognised by a T cell.
• an epitope For a given immunogenic region, it is typically possible to generate a "nested set" of overlapping peptides which act as epitopes, all of which contain the minimal epitope but differ in their flanking regions.
• T cell combination by measuring the response to truncated peptides. For example, if a response is obtained to the peptide comprising residues 1-15 in the overlapping library, sets which are truncated at both ends (i.e. 1-14, 1-13, 1-12 etc. and 2-15, 3-15, 4-15 etc.) can be used to identify the minimal epitope.
• the present inventors have previously determined that there is a link between the capacity of a peptide to bind to an MHC molecule and be presented to a T cell without further processing, and the peptide's capacity to induce tolerance in vivo (WO 02/16410). If a peptide is too long to bind the peptide binding groove of an MHC molecule without further processing (e.g. trimming), or binds in an inappropriate conformation then it will not be tolerogenic in vivo. If, on the other hand, the peptide is of an appropriate size and conformation to bind directly to the MHC peptide binding groove and be presented to a T cell, then this peptide can be predicted to be useful for tolerance induction.
• S-Ag apitopes (Antigen Processing-Independent epiTOPES) are capable of binding to a MHC class II molecule and stimulating a response from S-Ag specific T cells without further antigen processing.
• Such apitopes can be predicted to cause tolerance to S-Ag, following the rule-based method described in WO 02/16410.
• Peptides that bind to MHC class I molecules are typically 7 to 13, more usually 8 to 10 amino acids in length.
• the binding of the peptide is stabilised at its two ends by contacts between atoms in the main chain of the peptide and invariant sites in the peptide-binding groove of all MHC class I molecules. There are invariant sites at both ends of the groove which bind the amino and carboxy termini of the peptide. Variations in peptide length are accommodated by a kinking in the peptide backbone, often at proline or glycine residues that allow flexibility.
• Peptides which bind to MHC class II molecules are typically between 8 and 20 amino acids in length, more usually between 10 and 17 amino acids in length, and can be longer (for example up to 40 amino acids).
• peptides lie in an extended conformation along the MHC II peptide-binding groove which (unlike the MHC class I peptide-binding groove) is open at both ends.
• the peptide is held in place mainly by main-chain atom contacts with conserved residues that line the peptide-binding groove.
• the peptide derived from S-Ag is capable of binding to an MHC class II molecule without further processing.
• the peptide of the present invention may comprise all or a portion of the S-Ag-derived peptides shown as SEQ ID NOs 1-46.
• portion refers to a peptide that is derived from SEQ ID NOs 1-46 and contains at least a minimal epitope, that is, the peptide is capable of binding to the peptide-binding grove of an MHC class II molecule, being recognised by a T cell and inducing tolerance.
• Solubility may be an important consideration in peptide-mediated tolerance induction.
• solubility may be improved by incorporation of additional amino acids which may be Glycine (G), Lysine (K) and/or Glutamic acid (E) at both N and C termini.
• additional amino acids may be Glycine (G), Lysine (K) and/or Glutamic acid (E) at both N and C termini.
• a peptide according to the invention may have, for example, one, two or three additional amino acids at the N and/or C termini.
• the additional amino acids may be selected from Glycine (G), Lysine (K) and/or Glutamic acid (E). Different combinations of these amino acids may be added to the peptides according to the invention.
• a peptide according to the invention may have one, two or three Lysine (K) residues at both N and C termini.
• a peptide according to the invention may have one, two or three Glycine (G) residues at both N and C termini.
• a peptide according to the invention may have one, two or three Glutamic acid (E) residues at both N and C termini.
• a peptide according to the invention may have one Glycine and one Lysine residue at both N and C termini. In one aspect a peptide according to the invention may have one Glycine and two Lysine residues at both N and C termini.
• a peptide according to the invention may have one Glutamic acid and one Lysine residue at both N and C termini.
• a peptide according to the invention may have one Glutamic acid and two Lysine residues at both N and C termini.
• the peptide may have a Glycine spacer at both ends, followed by combinations of two additional amino acids which may be Lysine (K) and/or Glutamic acid (E) at both N and C termini.
• the possible combination at a given terminus may therefore be GKK, GKE, GEK or GEE.
• the peptide may have the general formula:
• a peptide according to the invention may have three additional Lysine (K) residues at both N and C termini.
• Modified peptides according to the present invention may therefore have 6 additional amino acids (3 at each end) than the parent peptides.
• the peptides of the present invention may alternatively have the general formula:
• the modified peptide may be more soluble that the parent (unmodified) peptide.
• the modified peptide may have 2, 3, 4, or 5-fold greater solubility than the parent peptide.
• the peptide may be soluble at concentrations of up to 0.5 mg/ml, 1 mg/ml, or 5 mg/ml.
• the peptide may be a modified form of one of the following peptides:
• VIFKKISRDKSVTIYLG SEQ ID NO: 14
• the modified peptides of the present invention may have 2, 4 or 6 additional amino acids (1 , 2 or 3 at each end) than the parent peptides.
• the modification is the inclusion of KKK at both the N and C termini.
• the modified peptides may have the sequence:
• the modified peptide may be more soluble that the parent (unmodified) peptide.
• the modified peptide may have 2, 3, 4, or 5-fold greater solubility than the parent peptide.
• the peptide may be soluble at concentrations of up to 0.5 mg/ml, 1 mg/ml, 5 mg/ml or higher, for example 8 mg/ml. In one aspect the modified peptide may be soluble at a concentration of 4mg/ml.
• S-arrestin also known as retinal arrestin, S-antigen or S-Ag
• S-arrestin is a soluble photoreceptor protein expressed in the retina and the pineal gland. It is known to be involved in desensitization of the photoactivated transduction cascade, and was first isolated from its binding to activated rhodopsin.
• the crystal structure shows two domains of anti-parallel ⁇ -sheets joined by a hinge region as well as a short a-helix at the back of the amino terminal fold.
• Rh* The light-activated form of the visual pigment rhodopsin (Rh*) interacts with the retinal G protein transducin, thereby initiating the exchange of a GDP molecule for GTP at the alpha- subunit of transducin.
• transducin In its GTP-binding form transducin dissociates from Rh*, and activates a cyclic GMP phosphodiesterase (PDE), by binding to its two inhibitory subunits PDEv.
• PDE cyclic GMP phosphodiesterase
• the turnover number for PDE can be in the order of several thousand hydrolysed cGMP per PDE per second.
• a rapid and effective elimination of Rh* is essential, before it can activate too many PDE molecules.
• This inactivation of Rh* is accomplished in two steps: phosphorylation of Rh* reduces its ability to catalyse the nucleotide exchange of transducin and subsequent binding of arrestin to P-Rh* completely shields it from further interaction with transducin.
• amino acid sequence of mature human S-Ag is given below (SEQ ID No.47).
• VDPDLVKGKK VYVTLTCAFR YGQEDIDVIG LTFRRDLYFS RVQVYPPVGA
• uveitis is commonly classified as one of the following based on the part of the eye which is primarily affected: anterior uveitis, intermediate uveitis, posterior uveitis or panuveitis .
• Anterior uveitis is the most common form of uveitis, and includes iridocyclitis and ulceris. Iritis is the inflammation of the anterior chamber and iris, while iridocyclitis includes inflammation in the ciliary body. Intermediate uveitis (pars planitis) commonly refers to vitritis - inflammation of cells in the vitreous cavity, associated with deposition of inflammatory material on the pars plana.
• Posterior uveitis is the inflammation of the retina and choroid regions.
• Panuveitis uveitis is a general term referring to inflammation affecting all layers of the uvea.
• Uveitis can also be classified as either infectious or non-infectious, with uveitis related to autoimmune diseases (i.e. primarily non-infectious) being more common in developed countries.
• the common animal models used to study uveitis are also driven by autoimmunity, showing a clear association between the two. It is predicted that 25-30% of uveitis is associated with systemic autoimmune or autoinflammatory diseases.
• the uveitis is non-infectious uveitis.
• T cell epitopes play a central role in the adaptive immune response to any antigen, whether self or foreign.
• the central role played by T cell epitopes in hypersensitivity diseases has been demonstrated through the use of experimental models. It is possible to induce autoimmune or allergic diseases by injection of synthetic peptides (based on the structure of T cell epitopes) in combination with adjuvant.
• Tolerance is the failure to respond to an antigen. Tolerance to self antigens is an essential feature of the immune system, when this is lost, autoimmune disease can result.
• the adaptive immune system must maintain the capacity to respond to an enormous variety of infectious agents while avoiding autoimmune attack of the self antigens contained within its own tissues. This is controlled to a large extent by negative selection of high-affinity T lymphocytes in the thymus (central tolerance). However, not all self antigens are expressed in the thymus, so death of self-reactive thymocytes remains incomplete. There are thus also mechanisms by which tolerance may be acquired by mature self-reactive T lymphocytes in the peripheral tissues (peripheral tolerance). A review of the mechanisms of central and peripheral tolerance is given in Anderton et al (1999) Immunological Reviews 169: 123-137. See also Wraith (2016) Nature 530:422-423.
• composition of the present invention is capable of inducing tolerance to self-antigens such as S-Ag, such that when administered to a subject, it may reinstate tolerance to the S-Ag protein and curtail the pathogenic immune response.
• composition of the present invention may be for prophylactic or therapeutic use.
• the composition When administered for prophylactic use, the composition may reduce or prevent the generation of an immune response to S-Ag.
• the level of immune response is less than would be obtained if the patient had not been treated with the composition.
• reduce indicates that a partial reduction in immune response is observed, such as a 50%, 70%, 80% or 90% reduction in the response that would have been observed if the patient had not been treated with the composition (or in the response observed in an untreated patient over the same time-period).
• prevent indicates that no appreciable immune response to S-Ag is observed.
• the composition When administered for therapeutic use, the composition may suppress an already on-going immune response to S-Ag.
• the term "suppress” indicates a reduction in the level of an on- going immune response, compared to the level before peptide treatment, or the levels which would have been observed at the same time point had the treatment not been given.
• Treatment with the composition of the present invention may cause a reduction in level of any or all of the following:
• B cells secreting S-Ag autoantibodies Detection of all of the factors can be carried out by techniques known in the art, such as ELISA, flow cytometry etc.
• Treatment with the composition of the present invention may also or alternatively cause anergy in CD4+ T cells specific for S-Ag. Anergy can be detected by, for example, subsequent challenge with S-Ag in vitro.
• Treatment with the composition of the present invention may cause generation of antigen-specific regulatory T cells, for example characterised by transcription factors c-Maf and NFIL3, and negative co-stimulatory molecules LAG-3, TIGIT, PD-1 and TIM-3 (see Burton et al. Nature Communications (2014) Article number 4741).
• composition may by prepared as an injectable, either as liquid solution or suspension; solid form suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
• the preparation may also be emulsified, or the peptides encapsulated in liposomes.
• the peptide may alternatively be encapsulated in a carrier or bound to the surface of a carrier, for example a nanoparticle.
• the active ingredients may be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline (for example, phosphate-buffered saline), dextrose, glycerol, ethanol, or the like and combinations thereof.
• the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents and/or pH buffering agents.
• Buffering salts include phosphate, citrate, acetate.
• Hydrochloric acid and/or sodium hydroxide may be used for pH adjustment.
• disaccharides may be used such as sucrose or trehalose.
• the relative ratio of the peptides may be approximately 1 : 1.
• the relative ratios of each peptide may be altered, for example, if it is found that one peptide works better than the others in particular HLA types.
• the composition may be incorporated into a sterile container which is then sealed and stored at a low temperature, for example 4°C, or it may be freeze-dried.
• composition is prepared as a lyophilised (freeze-dried) powder. Lyophilisation permits long-term storage in a stabilised form. Lyophilisation procedures are well known in the art, see for example http://www.devicelink.com/ivdt/archive/97/01/006.html. Bulking agents are commonly used prior to freeze-drying, such as mannitol, dextran or glycine.
• the composition may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intramuscular, subcutaneous, sublingual, intranasal, intradermal or suppository routes or implanting (e.g. using slow release molecules).
• composition may advantageously be administered via intranasal, subcutaneous or intradermal routes.
• the peptide or composition as described herein is typically administered in an "effective amount"; that is, an amount effective to elicit any one or more inter alia of a therapeutic or prophylactic effect.
• an effective amount that is, an amount effective to elicit any one or more inter alia of a therapeutic or prophylactic effect.
• Persons skilled in the art would be able, by routine experimentation, to determine an effective, non-toxic amount to include in a pharmaceutical composition or to be administered for the desired outcome.
• the peptide or composition as disclosed herein can be administered in a manner compatible with the route of administration and physical characteristics of the recipient (including health status) and in such a way that it elicits the desired effect(s) (i.e. therapeutically effective and/or protective).
• the appropriate dosage of a composition may depend on a variety of factors including, but not limited to, a subject's physical characteristics (e.g., age, weight, sex), and other factors that may be recognized by persons skilled in the art.
• a subject's physical characteristics e.g., age, weight, sex
• Other illustrative examples of general considerations that may be considered when determining, for example, an appropriate dosage of the compositions are discussed by Gennaro (2000, “Remington: The Science and Practice of Pharmacy", 20th edition, Lippincott, Williams, & Wilkins; and Gilman et al., (Eds), (1990), “Goodman And Gilman's: The Pharmacological Bases of Therapeutics", Pergamon Press).
• the peptide and composition of the invention may be used to treat a human subject.
• the subject may have uveitis.
• the subject may have S-Ag autoreactive T cells.
• the subject may be an HLA-haplotype which is associated with a predisposition to produce excessive T cells specific for S-Ag.
• Methods for determining the HLA haplotype of an individual are known in the art.
• the subject has an HLA gene selected from the following: A29, B51 , B27, DR8, DR4, DP5, DR4, DQA3, DR3, DR2, DR51 , and DR17 (see Mattapallil et al. J Immunol 201 1 , 187: 1977-1985).
• a "dose escalation" protocol may be followed, where a plurality of doses is given to the patient in ascending concentrations.
• Peptides derived from S-Ag may be administered together, in the form of a mixed composition or cocktail. However, there may be circumstances in which it is preferable to provide the peptides separately in the form of a kit, for simultaneous, separate, sequential or combined administration.
• the kit may comprise the peptides in separate containers.
• the contents of the containers may or may not be combined prior to administration.
• the kit may also comprise mixing and/or administration means (for example a vapouriser for intranasal administration; or a syringe and needle or other medical device for subcutaneous/intradermal dosing).
• administration means for example a vapouriser for intranasal administration; or a syringe and needle or other medical device for subcutaneous/intradermal dosing.
• the kit may also comprise instructions for use.
• the pharmaceutical composition or kit of the invention may be used to treat and/or prevent a disease, such as uveitis as discussed herein.
• composition/kit may be used to suppress or prevent the production of S-Ag- specific CD4+ T cells (or S-Ag autoantibodies) in vivo.
• the composition/kit may be used to treat and/or prevent uveitis in a subject.
• Model for analysing in vivo peptide presentation The present invention also encompasses a model for in vivo peptide presentation. As demonstrated in the present Examples (see e.g. Figure 14),
• the invention encompasses a method for ex vivo determination of in vivo peptide presentation, wherein said method comprises:
• CD4 + T cell activation and/or proliferation may indicate presentation of the peptide by an MHC molecule.
• analysis of CD4 + T cell activation is performed by assessing the level of IFN- ⁇ in the supernatant.
• Methods for analysing the level of IFN- ⁇ are known in the art, for example, ELISA methods.
• the mouse is a DR3tg or DR2tg mouse. In one aspect the mouse has been injected with about 100 ⁇ g of peptide, for example by subcutaneous injection. In one aspect the CD11c + cells are harvested from the spleen of the mouse.
• Allele frequencies were calculated from the allele frequency database (http://www.allelefrequencies.net).
• the allele frequency database consist of several thousand individual studies from various geographic locations and ethnic populations, with each study focusing on one specific ethnic or geographic population. The size of the cohort analysed and location varies greatly between studies, however the largest studies originate from Europe and North America.
• the frequencies in a general World Population were calculated as an average of allele frequencies from the individual studies weighted by the number of test-subjects. To avoid bias towards large studies, the number of test subjects in studies with more than 5000 was set to 5000. This ensures a lower bias in the region specific population, however the world average is still biased towards the European and the North American population.
• NetMHCII and NetMHCIIpan were employed for identification of potential T-helper cell epitopes for the most prevalent HLA-alleles, i.e. Alleles found in more than 1 % of the population.
• NetMHCII and NetMHCIIpan predicts interaction between MHC Class II molecules, encoded by the HLA loci in humans, and protein sub-15- mer.
• the tools are developed based on thousands of peptide-MHC interactions measurements and predicts IC50 values for a given 15-mer peptide and HLA-allele. However, a %Rank value instead of the predicted IC50 values was used to standardize predictions across MHC alleles as recommended by the IEDB (www.iedb.org).
• the %Rank value indicates the significance of the interaction value compared to interaction of random human derived peptides i.e. a %Rank value of 10 means that the interaction value is in the top 10% of the strongest measured interactions. The lower the rank values the higher the likelihood of a peptide binding to the given MHC molecule.
• NetMHCII2-2 for DP and DQ
• NetMHCIIpan-2.1 were used as described in Nielsen et al. (2010) Immunome Res 6, 9 and Nielsen and Lund (2009) BMC Bioinformatics 10, 296.
• Each protein sequence was analysed for sub-15mer MHC II binding peptides restricted to each of the HLA alleles in Table 4 by decomposing the protein to overlapping 15-mer peptides.
• a score for each sub-15mer peptide was calculated as the summed population frequency of HLA molecules predicted to bind the given peptide (Equation 1). This score is referred to as risk score, however it reflects the population coverage of HLA molecules predicted to bind to each sub-15mer peptide, and thus does not reflect clinical immunogenicity.
• tSqis tson 1 Risk score
• S P is the 15-mer peptide starting at position p
• r p the predicted rank score of the peptide starting at position p binding to allele a.
• f a is the allele frequency for allele a
• T epi is the binding threshold for considering a peptide a potential epitope (i.e. 10% rank).
• the maximum value of the risk score depends on the coverage of HLA alleles of the investigated population. However the theoretical max is 1 , which is achieved when all overlapping 15- mers for a given position are predicted to be bind to S P .
• SEQ ID Nos: 1-9 were found through evaluating the most potent potential epitopes, defined as having a risk score above 0.4.
• a risk score of 0.4 corresponds to approximately two standard deviations above the mean risk value for S-Ag.
• Apitopes are identified within the identified regions.
• solubility of potential apitopes from S-Ag are determined by visual observations and turbidity measurement by UV spectrophotometry at 280 nm.
• the solubility of the peptides can be modified by adding amino acids 'GKK' or 'KKK' (or similar) on both the C- and N- terminus of the sequence. Modifications that may be suitable are described in WO2014/072958 (which is hereby incorporated by reference).
• mice are pretreated with different apitopes according to the following schedules: Mice are injected subcutaneously in the flanks with S-Ag peptides (100 ⁇ g/injection) or PBS at day -8, -6, -4 (high dose schedule). Alternatively, mice are injected using a dose escalation schedule, wherein e.g. X ⁇ g of peptide are administered, followed by 10X ⁇ g, then 100X ⁇ g then 1000X ⁇ g.
• mice may be injected subcutaneously in the base of the tail with 50-100 ⁇ g antigen/CFA (S-Ag or the native sequence of the tolerogenic peptide).
• CFA antigen/CFA
• the draining LNs and spleens are harvested.
• Proliferation assay are then performed as described below.
• 60 ⁇ _ of cell supernatant are harvested and frozen.
• 20 ⁇ _ ⁇ / ⁇ of tritiated thymidine (PerkinElmer, Zaventem, Belgium) are then added to the cells to obtain a final concentration of 1 ⁇ / ⁇ .
• the cells are incubated at 37°C, and after 16h, plates are frozen. Thawed plates are harvested and read with ⁇ -counter (Wallac 1450 Microbeta Trilux Liquid Scintillation Counter) to assess the cell proliferation.
• SAg S-arrestin
• mice DR3tg mice were bred under specific pathogen-free conditions externally at Charles River, UK, or at Innoser, Netherlands.
• the DR3tg strain was originally created by Strauss et al (Strauss et al, 1994, Immunogenetics 3, 104-108).
• the genomic constructs used were a 6 kb Ndel fragment of a HLA-DRA genomic clone in pUC 13 and a 24 kb ClalxSall fragment of cos 4.1 , a cosmid (pTCF) containing the B gene of DRB1*0301.
• a solution containing 1-2 ⁇ g/mL of each construct was used for co-injection into fertilised eggs from (C57BL/6 x DBA/2) F1 donors mated with C57BL/6 males.
• the offspring were later bred into the IA-beta knockout B6; 129S2-H2dlAb1-Ea/J lacking mouse MHC class II molecule expression.
• These DR3tg mice express the HLA-DRB1*0301 molecule but not the mouse MHC-II molecule.
• the mice were maintained by backcrossing to C57BL/6 and to B10.Q.
• mice were identified by Southern blot analysis of tail DNA digested with EcoR and probed with a 1.35 kb BamH ⁇ fragment of the DRA cDNA and a 1.25 kb BamH ⁇ fragment of the DRB1*0301 cDNA.
• DR3tg mice were used for these experiments as it has been suggested that this MHC class II molecule is associated with an increased risk for individuals to develop uveitis disease.
• DR2tg mice were bred under specific pathogen-free conditions externally at Charles River, UK, or at Innoser, Netherlands.
• HLA-DR2 transgenic (DR2tg) mice were originally obtained from Lars Fugger (Madsen et al., 1999).
• DRa and DR ⁇ chain cDNAs were expressed by using the pDOI-5 expression vector which contains a mouse MHCII promotor.
• the constructs were injected into fertilised eggs from (DBA/2xC57BL/6)F1 matings.
• the mice were backcrossed into the IA-beta knockout C57BL/6 genetic background (ABO mice) lacking mouse MHC class II molecule expression.
• the DR2tg mice express the HLA-DRB1*1501 molecule but not the mouse MHC molecule.
• 5x10 4 cells from the individual clones were cultured with 10 ⁇ g/ml peptide and 5x10 4 fixed or fresh APCs.
• 5x10 4 cells were cultured with 10 ⁇ g/ml and 25 ⁇ g/ml peptide and 2.5x10 4 fixed or fresh APCs.
• To fixate APCs cells were incubated with 0.5% paraformaldehyde (Merck, Darmstadt, Germany) (pH7) for 5 min at room temperature (RT).
• the fixation reaction was stopped by adding 0.4M glycine (Sigma-Aldrich) and washing the cells in RPMI-10% FCS. Additionally, reactivity towards human SAg protein (QBiologicals, Eurofins Amatsigroup, Ghent, Belgium) was measured to identify cryptic epitopes. After 24h or 48h, antigen- induced IFN- ⁇ or IL-2 production (respectively) was measured by ELISA (R&D Systems, Abingdon, UK).
• DR3tg or DR2tg mice were injected subcutaneously in the flank region with 0.1 ⁇ g, 1 ⁇ g and 10 ⁇ g of peptide on days -15, -13 and -1 1 respectively, followed by 3 injections of 100 ⁇ g peptide on days -8, -6 and -4 (dose escalation schedule).
• the mice were immunised subcutaneously in the base of the tail with 50 antigen (parental peptide) emulsified in CFA (peptide/CFA).
• CFA peptide/CFA
• LN cells and splenocytes were isolated and cultured in X-vivo 15 medium (supplemented with 2mM L-glutamine, 50 U/mL penicillin and 50 U/mL streptomycin; Lonza) in 96-well flat bottom plates.
• X-vivo 15 medium supplemented with 2mM L-glutamine, 50 U/mL penicillin and 50 U/mL streptomycin; Lonza
• 0.5x10 6 cells/well were cultured (200 ⁇ /well) for 72 hours with different antigen concentrations (0-25 ⁇ g/ml) or with 12.5 ⁇ g/ml purified protein derivative (PPD; priming control; Statens serum institut, Copenhagen, Denmark). After 72 hours, supernatant was harvested and stored at -80°C until further analysis. IFN- ⁇ concentrations in supernatants were assessed by cytokine ELISA (R&D Systems, Abingdon, UK) to measure cell activation.
• TCL Tcell line
• DR3tg or DR2tg mice were immunised subcutaneously in the base of the tail with 50 ⁇ g peptide emulsified in CFA (peptide/CFA).
• CFA peptide/CFA
• LN cells and splenocytes were isolated and CD4 + T cells were isolated by negative selection using Magnisort Mouse CD4 Isolation kit (ThermoFisher Scientific) according to the manufacturer's instructions. Irradiated (3000 rad) splenocytes were used as antigen-presenting cells (APC).
• APC + 2.5x10 6 CD4 + T cells were cultured in X-vivo 15 medium (supplemented with 2mM L-glutamine, 50 U/mL penicillin and 50 U/mL streptomycin; Lonza) in 6-well plates in the presence of 0, 1 ; 1 ; 2,5 or 5 ⁇ g/ml of peptide.
• 20U/ml of rlL-2 (R&D Systems, Abingdon, UK) was added.
• TCL cells were counted and cultured with fresh APC, peptides and IL-2, all at the same concentrations as above.
• 20 U/ml of rlL-2 was added.
• TCL cultures were used as such, or CD4 + cells were selected.
• DR3tg or DR2tg mice were injected with 100 ⁇ g of peptide in 100 ⁇ PBS subcutaneously (s.c.) in the flank. Control animals received a s.c. injection of 100 ⁇ PBS. After 2 hours, spleens were harvested and single-cell suspensions were made. CD1 1c + cells were positively selected using CD1 1c microbeads according to the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Average purities of >92% were reached.
• CD11 c + cells were co-cultured with 5x10 4 CD4 + cells in round bottom 96-well plates in X-vivo 15 medium (supplemented with 2mM L-glutamine, 50 U/mL penicillin and 50 U/mL streptomycin; Lonza). These CD4 + cells were isolated from peptide-specific T cell lines (see above) by positive selection using CD4 microbeads according to the manufacturer's instructions (Miltenyi Biotec). After 48 hours, supernatant of these co-cultures was collected and CD4 + T cell activation was analysed by IFN- ⁇ ELISA (R&D Systems, Abingdon, UK). In a parallel experiment, CD4 + T cell responses towards peptide added in vitro were assessed to make sure the T cells recognize the peptides presented by the CD11 c + cells.
• Apitopes are capable of binding to an MHC molecule and stimulating a response from SAg specific T cells without further antigen processing.
• Such apitopes can be predicted to cause tolerance to SAg, following the rule- based method described in WO 02/16410.
• Figures 2 to 6 show the identification of apitopes within the sequences that were previously identified as immunogenic.
• Figure 2 focuses on the HIP-1 15 region (SEQ ID No 1).
• the ability of both a hybridoma clone (Fig. 2A) and a T cell line (Fig. 2B) to respond to peptides that were picked up and presented by fresh or fixed antigen presenting cells (APC) indicates that HIP-115, HIP- 115NE, HIP-1 15NE1 , HIP-1 15NE2, HIP-115NE3 and HIP-115NE3-KKK behave as apitopes.
• Figure 3 focuses on the HIP-241 125 region (SEQ ID No 7). In this case, different apitopes were identified depending on whether the response to peptide was assessed using hybridomas (Fig. 3A) or T cell line (Fig. 3B). HIP-1 1AC, HIP-1 1AC1 and HIP-1 1 B behave as apitopes (Fig. 3A), and so do HIP-24DG, HIP-24HM, HIP-24HM2, HIP-24HM3 and HIP- 241125 itself.
• Figure 4 focuses on the HIP-9FL region (SEQ ID No 9). Peptides HIP-9FL, HIP-9FL-KKK and HIP-9K1-KKK behave as apitopes.
• Figure 5 focuses on the HIP-17GN region (SEQ ID No 3). Peptides HIP-17GN, HIP-17GN- KKK, HIP-17J, HIP-17J-KKK and HIP-17K1-KKK behave as apitopes.
• Figure 6 focuses on the HIP-12AK region (SEQ ID No 8).
• Peptides HIP-12G1 , HIP-12G1- KKK and HIP-12E-KKK behave as apitopes. 3. 7 cell tolerance induction by single peptides
• transgenic mice were treated according to the dose escalation schedule ( Figure 7) with each one of the peptides.
• the parental peptides were insoluble, and solubility is a critical parameter for them to work as a tolerogenic peptide.
• end-terminal modifications can be used and as example used here they were improved by addition of 3 lysines before and after the original peptide sequence (labelled as '-KKK' versions).
• Figure 8 shows how the soluble form of peptide HIP-115NE3 (HIP-1 15NE3-KKK) is capable of inducing tolerance against SAg in LN (Fig. 8A) and in spleen (Fig. 8B), amounting to 70% reduction in response to SAg when the highest dose of SAg was used for probing. Furthermore, HIP-1 15NE3-KKK induces tolerance against HIP-1 15 and HIP-115NE3 peptides in LN (Fig. 8C) and spleen (Fig. 8D); 55% and 65% reduction in response respectively at the highest concentrations of probing peptide.
• HIP-1 15NE3-KKK induces tolerance against HIP-1 15 and HIP-115NE3 peptides in LN (Fig. 8C) and spleen (Fig. 8D); 55% and 65% reduction in response respectively at the highest concentrations of probing peptide.
• Figure 9 shows the tolerization induced by peptide HIP-11 B against SAg in LN (Fig. 9A) and in spleen (Fig. 9B), and especially against HIP-11 B itself in LN (Fig. 9C) and in spleen (Fig. 9D).
• Figure 10 shows the tolerization induced by peptide HIP-24DG against SAg in LN (Fig. 10A). There is some tolerization against HIP-24DG itself (Fig. 10B) and other peptides within the same region, namely HIP-24HM2 (Fig. 10C) and HIP-24HM3 (Fig. 10D), all results shown for LN.
• Figure 1 1 shows the tolerization induced by peptide HIP-9K1-KKK in LN against SAg (Fig. 11 A). HIP-9FL and HIP-9K1-KKK (Fig. 1 1 B). Tolerance is also induced in spleens against SAg (Fig. 11 C) and to HIP-9K1-KKK itself (Fig. 11 D).
• Figure 12 shows the tolerization induced by peptides HIP-17GN-KKK and HIP-17J-KKK against SAg in spleen.
• Figure 13 shows the tolerization induced by peptide HIP-12G1-KKK against itself in LN (Fig. 13A) and in spleen (Fig. 13B). 4. In vivo presentation of single peptides
• HIP-1 15NE3 insoluble peptide
• HIP-115NE3-KKK soluble peptide
• DC dendritic cells
• HIP-115NE3-KKK is bound to the MHC II on DC in situ and presented to peptide- specific T cells after DC cell isolation. T cell activity response was measured by production of IFN- ⁇ .
• the insoluble peptide was not able to cause this activation showing solubility to be a critical property for tolerogenic peptides.
• HIP-24DG HIP-24HM2 and HIP-24HM3 peptides (all soluble) to be picked up and presented by MHC II molecules present on dendritic cells (DC) upon injection in an in vivo setting (DR3tg mice) was compared.
• HIP-24HM3 is presented by DC to T cells, which become activated and produce IFN- ⁇ .
• antigen processing independent epitopes (ii) can be presented ex vivo to peptide specific T cells by MHCII molecules present on dendritic cells and (ii) are able to induce tolerance against SAg or fragments of it when administered using the escalating dose regime of treatment.

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